Pure human renin. Identification and characterization and of two major molecular weight forms.
Human renal renin was purified from normal kidney by either of two protocols which combined sequential DEAE-cellulose chromatography, pepstatin affinity chromatography, gel filtration, and a final step of affinity chromatography using either the synthetic octapeptide renin inhibitor (D-Leu6] or antirenin immunoglobulin as ligand. An approximate 500,000-fold purification and a yield of 1 mg of protein or 7% enzymatic activity from 10 kg were obtained by either method. Maximum specific activity was 1170 Goldblatt units/mg. Amino acid composition and kinetic properties were determined. Using purified angiotensinogen substrate, optimum pH was 5.5-6.0 and the Km was 1.54 X 10(-6) M. Two major forms of renin possessing similar enzymatic and immunologic properties, but differing in apparent molecular size and charge were purified and characterized. One form, the major form obtained after antibody affinity chromatography, had an apparent molecular size of 50 kilodaltons by sodium dodecyl sulfate-gel electrophoresis and migrated more slowly (RF = 0.32) on polyacrylamide disc gel electrophoresis at pH 7.8. The other form had an apparent molecular size of 39 kilodaltons and migrated more rapidly (RF = 0.76) on polyacrylamide disc gels. This smaller form predominated in protocols which allowed the persistent presence of acid protease activity throughout purification. Moreover, renin molecular size was demonstrated to change from 50 to 40 kilodaltons in the presence of this protease, which was subsequently isolated from the penultimate step of renin purification and tentatively identified as a renal cathepsin D. These findings help reconcile certain disparate characteristics for pure human renin obtained by others, explain the marked instability of the human enzyme, and suggest that the apparent molecular size of human renin is somewhat larger than had been previously reported.
D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D005779:Immunodiffusion; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D012083:Renin / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D012083:Renin